Characterization of placental endocrine function and fetal brain development in a mouse model of small for gestational age

Abstract

Conditions such as small for gestational age (SGA), which is defined as birthweight less than 10^th percentile for gestational age can predispose to neurodevelopmental abnormalities compared to babies with normal birthweight. Fetal growth and birthweight depend on placental function, as this organ transports substrates to the developing fetus and it acts as a source of endocrine factors, including steroids and prolactins that are required for fetal development and pregnancy maintenance. To advance our knowledge on the aetiology of fetal growth disorders, the vast majority of the research has been focused on studying the transport function of the placenta, leaving practically unexplored the contribution of placental hormones in the regulation of fetal growth. Here, using mice and natural variability in fetal growth within the litter, we compared fetuses that fell on or below the 10^th percentile (classified as SGA) with those that had adequate weight for their gestational age (AGA). In particular, we compared placental endocrine metabolism and hormone production, as well as fetal brain weight and expression of developmental, growth and metabolic genes between SGA and AGA fetuses. We found that compared to AGA fetuses, SGA fetuses had lower placental efficiency and reduced capacity for placental production of hormones (e.g. steroidogenic gene Cyp17a1, prolactin Prl3a1, and pregnancy-specific glycoproteins Psg21). Brain weight was reduced in SGA fetuses, although this was proportional to the reduction in overall fetal size. The expression of glucose transporter 3 (Slc2a3) was reduced despite the abundance of AKT, FOXO and ERK proteins were similar. Developmental (Sv2b and Gabrg1) and microglia genes (Ier3), as well as the pregnancy-specific glycoprotein receptor (Cd9) were lower in the brain of SGA versus AGA fetuses. In this mouse model of SGA, our results therefore demonstrate that placental endocrine dysfunction is associated with changes in fetal growth and fetal brain development.

Keywords: animal models; endocrinology; fetal growth; mouse; placenta; pregnancy; pregnancy specific glycoprotein; prolactin.

Figure 1

Fetal and placental weight in mice and establishment of a model to study small for gestational age. (A) Frequency distribution curve for fetal weights on GD16. Vertical line denotes the 10^th (321mg) and 90^th percentiles (410 mg). (B) Fetal weight for fetuses classified as SGA, AGA and LGA. (C) Placental weight classified as SGA, AGA and LGA. (D) Placental efficiency (defined as grams of fetus produced by grams of placenta) classified as SGA, AGA and LGA. (E–G) Feto-placental weights and placental efficiency based on their position in the uterus. (H) Distribution of LGA, AGA and SGA fetuses according to their position in the uterus. (I–K) Feto-placental weights and placental efficiency based on litter size. (L) Distribution of LGA, AGA and SGA fetuses according to litter size. Data are from 90 fetuses in total from 11 litters. Feto-placental weights are shown as box plots and whiskers. The rectangle shows the distribution, the line the median, the whiskers the maximum and minimum and the “+” is the mean of the group. Statistical analysis performed by one-way ANOVA, Student t-test, Mann-Whitney test and Chi-square test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AGA (appropriate for gestational age), LGA (large for gestational age), SGA (small for gestational age), n° (number).

Figure 2

Small for gestational age is associated with altered expression of placental hormones. (A, B) Immunoblots and protein abundance of key growth and metabolic signalling proteins in dissected placental junctional zones (n=5 samples per group). (C) Expression of key genes involved in placental function and growth (n=9 samples per group). (D–F) Expression of steroidogenic pathway regulatory (D), pregnancy specific glycoprotein (E) and prolactin (F) genes in dissected placental junctional zones (n=9 samples per group). (G) Heatmap of Pearson’s correlations (number inside the box corresponds to the r value for the correlations that were significant) (n=18 samples). Data are shown as individual values and bars represent mean ± SEM. Statistical analysis performed by paired or unpaired t-test (Student t-test and Mann-Whitney test, respectively). *P<0.05, **P<0.01. AGA (appropriate for gestational age) and SGA (small for gestational age), FW (fetal weight), FP (feto-placental ratio, also known as placental efficiency), PW (placental weight), P (phosphorylated protein levels), T (total protein levels).

Figure 3

Small for gestational age and its impact on fetal brain. (A) Brain weight. (B) Brain weight expressed as a ratio of the brain weight divided by fetal weight. (C) Brain weight expressed as a ratio of brain weight divided by placental weight. (D) The expression of key prolactin and pregnancy-specific glycoprotein receptors in fetal brain (n=8-9 samples per group). (E, F) Immunoblots and protein abundance of key growth and metabolic signalling proteins in fetal brain (n=5 samples per group). (G–I) Expression levels of glucose transporters, microglia and axon developmental genes (n=8-9 samples per group). Data are from 90 fetuses in total from 11 litters (excluding outliers). Data are shown as individual values and bars represent mean ± SEM. Statistical analysis performed by one-way ANOVA followed by Bonferroni post hoc test (variables of three groups), paired or unpaired t-test (Student t-test and Mann-Whitney test, respectively). ROUT test was conducted to detect and eliminate outliers. *P<0.05, **P<0.01, ***P<0.001. AGA, (appropriate for gestational age); SGA, (small for gestational age); P, (phosphorylated protein levels); T, (total protein levels).